This invention relates to the use of polymers or biopolymers for the preparation of a drug for the treatment of lesions of all origins affecting the central or peripheral system in human or veterinary medicine. It also concerns pharmaceutical compositions for this treatment. Lastly, it concerns fractions enriched in heparan sulfate.
The synthesis of CMDBS polymers (dextrans substituted by carboxymethyl, benzylamine and sulfonate) has been described in French Patent 2 461 724 and U.S. Pat. No. 4,740,594. Some of these polymers mimic heparin and may be used as plasma heparin replacement products, thanks to their anticoagulant and anticomplement properties.
Some of these CMDBS polymers mimic another property of heparin consisting of a stabilization, protection and potentialization of the in vitro biological activity of the growth factors of the FGF family (Tardieu and coll., Journal of Cellular Physiology, 1992, 150 pp. 194 to 203).
French patent 2 644.066 describes the use of certain CMDBSs associated with FGFs for healing the skin and the cornea. Experiments have been conducted by provoking a cutaneous wound with the help of a hollow punch 6 mm in diameter in the rat. In this example, the CMDBS associated with the FGF 2 makes it possible to obtain a clear effect on the speed and the quality of skin repair.
Another biopolymer, dektran sulfate, has also been advanced in association with FGFs as a stabilizer and protector, in Japanese Patent No. 13890. Dextran sulfate, moreover, is widely used in skin healing ointments and creams as well as in collyrium compositions, but, to the knowledge of the applicant, has no reported effect on the healing and regeneration of lesions of the nervous system.
A considerable number of factors whose activities facilitate the survival of nervous cells, the repair of central or peripheral nervous lesions, or muscular reinnervations have been described: polypeptide factors like Nerve Growth Factors (NGF), factors of the FGF families, brain derived factors like BDNFs, Ciliary Neurotrophic Factor (CNTF), Neurotrophin 3 (NT3), etc.
These factors have been used in experiments of muscular reinnervation, repairs of cuts of peripheral nerves, motor nerves, in models of lesions of cholinergic central nervous cells, and in numerous other models. For purposes of reference, the reviews listed below describe a part of this work: P. M. Richardson, Current Opinion in Neurobiology, 1991, 1: pp 401-406; T. Ebendal, Journal of Neurosciences Research, 1992, 32: pp. 461-470; P. G. Cordeiro, R. Brooke et al., Plastic and Reconstructive Surgery, 1989, 86 (3): pp. 1013-1019; Q. Yan, J. Elliott et al., Nature (Letters to Nature), 1992, 360: pp. 753-755; N. A. Seniuk, Journal of Reconstructive Microsurgery, 8 (5): pp. 399-404; F. Hefti P. P. Michel et al., Advances in Neurology, 1990, 53: pp. 123-127; A. C. Cuello, L. Garofalo et al., Progress in Brain Research, 1990, 84: pp. 301-311; A. Tadeka, H. Onodera et al., Brain Research, 1992, 569: pp. 177-180.
It thus emerges from an analysis of the state of the prior art that growth factors and polymers in association with growth factors have already been used in therapeutic applications.
However, none of the documents cited above shows that the polymers present effects by themselves, that is to say without being associated with growth factors.
Moreover, the activity of polymer-factor associations has been described only on certain lesions of a very specific type of tissue, namely the cutaneous tissue.
In view of the unpredictable nature of the therapeutic effects of a given molecule, it was not clear whether these polymers could have an effect on other tissues.
It is, in fact, well known that the different tissues of the human or animal body present structural and functional specific features making it impossible to predict the effect of a molecule, known for its effect on the cutaneous tissue or another tissue.
Similarly, it is well known that it is impossible to predict the in vivo activity of a molecule on a particular tissue from results obtained in vitro on a specific experimental model.
Surprisingly, it has been found, according to the invention, that certain polymers have a very marked effect on the speed of healing and of regeneration of tissue lesions of the central or peripheral nervous systems as well as on the quality of this healing and/or regeneration, in such a way that it may be measured by studying it using histological and physiological methods. Muscular reinnervation with reformation of a functional junction of the lesioned nerve with its muscle was observed.
This invention relates to the use of at least one polymer or one biopolymer, caller HBGFPP, with the exception of mesoglycan, specifically protecting the growth factors of the FGF and beta TGF families from tryptic degradation and not significantly inhibiting coagulation, in the manufacture of a drug for the treatment of muscular tissues.
In particular, such a polymer presents an anticoagulant activity of less than 50 international units per mg of polymer measured, according to Maillet et al. (Mol. Immunol, 1988, 25, 915-923). Preferentially, it does not substantially activate the complement system, that is to say, it possesses an anti-complement system of above 0.5 xcexcg for the CH50 (according to Mauzac et al., Biomaterials, 6, 61-63, 1985).
Advantageously, the polymer potentializes the FGFs in vitro.
According to the invention, polymers are understood to mean any natural substance, chemically modified natural substance or totally synthetic substance responding to the definition given above.
The following polymers are therefore concerned:
polymers obtained from dextrans but modified by other types of substitutions with other types of radicals,
natural polymers other than those deriving from dextrans but including osidic residues (cellulose, chitin, fucans, etc.),
polymers obtained by polymerization of monomers of non-osidic nature (modified or unmodified malic polyacid, oxalic polyacid, lactic polyacid, polystyrene, polyethylene glycol).
Advantageously, the said polymer or biopolymer is a polysaccharide which may be primarily composed of glucose residues.
Such a polysaccharide advantageously presents a molecular weight above 10 kD and advantageously about 40 kD.
It may also comprise glucosamine and/or uronic acid residues, particularly in the form of glucosamine dimer-uronic acid.
Particularly preferred polysaccharides are substituted dextrans, glycosaminoglycans possibly in association with a lipid, a peptide or a protide, or sulfates of these polymers.
This invention also relates to a pharmaceutical composition containing these polymers.
The polymers and/or biopolymers may be selected from natural substances which may then be modified, if required, by additions of appropriate chemical groups, or again be obtained entirely by synthesis. These natural, semi- or wholly synthetic polymers are then selected on the basis of their ability to interact specifically with several growth factors, notably those of the FGF and the beta TGF families. They are also selected on their ability to protect this (or these) factor(s) against proteolytic degradations. These polymers will be referred to under the generic abbreviation HBGFPP (heparin binding growth factor protectors and promoters).
Two prototypes of these polymers or biopolymers are given as examples together with the processes and selection criteria of these polymers.
The first HBGFPP example belongs to the CMDBS family which are known products, namely functionalized biospecific dextrans, substituted by carboxymethyl, benzylamide and benzylamine sulfonate. These polymers illustrate the yielding of HBGFPPs from natural products (dextrans) which are subsequently chemically substituted.
The second example describes the selection of wholly natural products such as purified sulfate proteoglycosaminoglycans from tissular extracts.
These two examples illustrate the ability of these HBGFPPs to interact, stabilize, protect and potentialize the growth factors of the FGF and beta TGF families, and their use in a pharmaceutical composition permitting a healing and a regeneration of nervous liaisons and a protection and healing of the nervous cells.
In this patent application, by  less than  less than treatment greater than  greater than  is meant any curative or preventive operation carried out for the prophylaxis, the healing, the protection or the regeneration of lesions touching the nervous system.
Thanks to the action of the HBGFPPs and in particular the CMDBSs, as the examples below illustrate, the reinnervation of the EDL or Soleus type muscle is accelerated. This reinnervation is manifested by the regeneration of the nervous fiber and the speedy reformation of a functional synaptic junction.
Not only is there an increase of axonal growth in lesions near the muscle but also a control of this growth. Thus, with respect to sprouting, this growth takes place in an organized and directed way resulting in an acceleration of functional repair.
The properties of HBGFPPs are such as to make this family of molecules a totally new and unique class of drugs which may be used to favor and improve lesions of the central or peripheral nervous system, directly touching the neuronal cells and their axonal and dendritic prolongations, cholinergic or dopaminergic neurons or again touching cells associated with neurons such as oligodendrocyte astrocyte glia cells and Schwann cells. These lesions may be of any origin: traumatic, iatrogenic or chemical, due to the use of radiation or induced by surgical operations, of bacterial, parasitic or viral infectious origins, of auto-immune origin, or lesions and deteriorations may be induced by bleedings such as ruptured vessels. These new drugs are also used in the treatment of neurodegenerative diseases such as Parkinson""s or Alzheimer""s disease, or of genetic origin. Lastly, these drugs may with benefit be associated with cells used in treatments of transplantations in the affected areas of the brain, using normal or genetically modified cells.
The drug and the pharmaceutical composition according to the invention may contain an effective quantity of HBGFPP, for example CMDBS associated with one or more compatible and pharmaceutically acceptable vehicles. It may also be associated with pharmaceutical agents such as anti-inflammatory agents and/or antibacterials.
The vehicle may be physiological serum or buffers such as PBS containing 0.15M NaCl or any other compatible solution which does not irritate the damaged nervous tissues. Formulations providing thick or gel solutions according to standard techniques known to the person of ordinary skill in the art may be proposed depending on the type and the accessibility of the lesion.
Advantageously, such a composition is designed to be injectable directly on the site of the lesion at a dose of 2.5 to 2500 mg/ml of HBGFPP as exemplified by CMDBS, or like natural HBGFPP biopolymers such as mesoglycan, but the intravenous or intramuscular route may be preferred in the case of de-innervation of de-innervated muscle. Injection in the spinal cord may also be preferable in the case of lesions of said spinal cord or of lesions of branches of prolongations of nervous fibers being motor and/or sensitive fibers. The injection volume is estimated in function of the size of the lesion. Doses corresponding to 100 xcexcl often prove sufficient.
In addition to the xe2x80x9cHeparin Bindingxe2x80x9d growth factor protection qualities, the HBGFPPs selected according to the tests described below present a very low anticoagulant activity compared to that of heparin, too weak to hamper coagulation in the case of a trauma. In the case of an injection by intravenous route, the injected dose must be adjusted to the blood volume of the man or animal treated in this way so that the dose of HBGFPP in the blood also lies between 2.5 and 2,500 mg/ml.
In the examples described in the following pages, concerning the reinnervation of the skeletal muscle of the male EDL-type rat (Extensor Digitorum Longus) or the postural slow muscles (soleus), a single injection of 100 xcexcl of a solution of CMDBS at 50 xcexcg/ml on the site of the wound induces a complete reinnervation in 17 days, whereas, in the case of the EDL muscle, this innervation is observed only after 60 days when no treatment is given. With regard to the soleus muscles, the CMDBS effect is even more pronounced since, after injection of CMDBS, reinnervation is total and functional in 17 days whereas the reinnervation of the controlateral muscle is defective even after 60 days. These effects are specific to the HBGFPPs and notably certain CMDBSs responding to selection criteria with respect to FGFs and beta TGF family growth factor protection against proteolytic degradations induced by the action of trypsin. This specificity may be illustrated by comparing the effects of repairing nervous tissue lesions by HBGFPPs and related products such as heparin, dextran, dextran sulfate or sucrase (sucrose octyl sulfate). Although these molecules interact with the FGFs, and at any rate as far as heparin is concerned with beta TGF, neither sucrase, nor heparin nor dextran sulfate protect the beta TGF against the proteolysis induced by the action of trypsin, as is shown by the application of screening and selection tests of the HBGFPPs described in the examples below. These products have no effect on lesions of the nervous tissues. Thus, by carrying out in vitro screening on the basis of a double protection of the FGFs and beta TGFs against the proteolysis induced by Trypsin, it is possible to select HBGFPPs, like certain CMDBSs including those given in these examples. These same selection criteria applied to natural biopolymers such as mesoglycan or sulodexide have shown that mesoglycan, which presents a double protection and stabilization activity for both FGFs and beta TGFs, has a beneficial activity in nervous repair and regeneration, and, as such, belongs to the HBGFPP family, whereas sulodexide, which protects the FGFs against the proteolysis induced by the activity of trypsin, has no significant protective action against the action of trypsin on beta TGFs.
This invention also relates to fractions of mesoglycan or sulodexide enriched in heparan sulfate, presenting advantageously 80%, and preferably 95%, of heparan sulfate.
Such fractions may be obtained by a heparan sulfate enrichment process of a glycosaminoglycan composition, comprising the following stages:
ion exchange chromatography of the composition,
elution on a DEAE gel,
treatment by ABC chondroitinase,
chromatography on molecular sieve, and
heparan sulfate elution.